This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-356047, filed Nov. 22, 2000, the entire contents of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates to a method of producing a biosensor protein capable of regulating a fluorescence property of green fluorescent protein. Furthermore, the present invention relates to a biosensor protein (hereinafter also referred to as a biosensor) produced by the above method and a biosensor gene encoding the biosensor protein. More specifically, the present invention relates to a calcium-sensing biosensor protein (hereinafter also referred to as a calcium sensor) produced by the above method and a biosensor gene encoding the calcium-sensing biosensor protein.
2. Description of the Related Art
Up to now, several biosensors have been developed by applying the fluorescence resonance energy transfer (FRET) to green fluorescent protein (hereinafter also referred to as xe2x80x9cGFPxe2x80x9d). Recently, it was reported that a new type of biosensor wherein a single GFP was capable of changing the fluorescence intensity without applying FRET has been developed. However, the sensitivity of the new type of biosensor was so low that it was not used in practice. To make the present invention, the present inventor has newly found that the problem of the low sensitivity was caused by insufficient examination of the linking site between GFP and a functional protein employed in the biosensor.
With respect to a calcium sensor among biosensors, roughly four types of calcium sensors have been developed. Hereinafter, outlines and shortcomings of these calcium sensors will be described.
1) A Calcium-Sensitive Synthetic Dye:
This is a chemically synthesized dye which has sensitivity to calcium, and widely used at present. The dye is loaded from the outside into a cell at the time of use. Membrane-permeable form of the dye (AM form) is easy to load into cells. However, AM form of the dye is loaded into all of the cell, so that it is difficult to introduce the dye into only a specific cell. To introduce the dye into the specific cell, the dye must be injected by the use of a glass needle or the like.
2) Aequorin:
This is a protein which reacts to calcium and emits light. Aequorin is directly injected into cells at the time of use, or an aequorin-encoding gene is incorporated into a cell before use. However, aequorin requires a coenzyme to function in a cell, so that the coenzyme must be supplied to the cell. Furthermore, the light emitted therefrom is extremely weak.
3) A Calcium-Sensitive Protein to which FRET is Applied:
This protein is composed of calcium-sensitive calmodulin (CaM), a partial sequence of myosin light chain kinase (which is bound to CaM), and two GFPs differing in fluorescence color. The protein utilizes the characteristics that the distance between two GFPs becomes shorter by a conformational change of CaM when calcium is bound to CaM, thereby inducing FRET, and as a result, the intensity of the fluorescence emitted from the two GFPs are altered. At the time of use, the protein is directly injected into cells, or the gene encoding the protein is incorporated into a cell. However, the change in the fluorescence intensity caused by the FRET is a little, and thus its signal is extremely weak. It is therefore hard to detect the signal by a conventional laser microscope equipped with an argon laser (xcex=488 nm).
4) A Calcium-Sensitive Protein Composed of a Single GFP:
The calcium-sensitive protein has structure where CaM is bound between the 144th and the 146th amino acids of the amino acid sequence of GFP. The protein utilizes the characteristics that the CaM protein causes a conformational change of the GFP when calcium is bound to CaM, thereby altering the intensity of the fluorescence emitted from GFP. This protein is directly injected into a cell at the time of use, or the gene encoding the protein is introduced into a cell before use. However, since the calcium sensitivity of the protein is low, so that the signal/noise ratio becomes low in the cell in fact. This fact makes it difficult to measure a signal.
The present invention was made with the view of overcoming the aforementioned problems. An object of the present invention is to provide a method of producing a biosensor protein capable of regulating a fluorescence property of green fluorescent protein, and to provide a biosensor protein produced by the method and a biosensor gene encoding the biosensor protein. Particularly, an object of the present invention is to provide a calcium-sensing biosensor protein produced by the method and a biosensor gene encoding the calcium-sensing biosensor protein.
More specifically, the object of the present invention is to produce a biosensor protein (particularly a calcium-sensing biosensor protein) whose sensitivity to a substance to be detected is high enough to use practically and which makes it possible to measure easily. Further object of the present invention is to produce a biosensor protein (particularly a calcium-sensing biosensor protein) which is introduced into a specific cell easily and which makes it possible to measure without a specific detection device, a coenzyme, and so on.
To attain the aforementioned objects, the present invention provides the means [1] to [19] described below.
[1] A method of producing a biosensor protein capable of regulating a fluorescence property of green fluorescent protein or its derivative by modifying the structure of green fluorescent protein or its derivative, comprising the steps of:
(A) predicting a hotspot amino acid residue affecting a fluorescence property of green fluorescent protein or its derivative;
(B) producing various fusion proteins which have the structure linked with a modified fluorescent protein and one or more functional molecules,
the modified fluorescent protein being the protein obtained by cleaving amino acid sequence of green fluorescent protein or its derivative in the vicinity of the hotspot amino acid residue and modifying the structure of green fluorescent protein or its derivative, and
the functional molecules each being the molecules capable of transmitting their conformational changes to the modified fluorescent protein to cause a conformational change of the modified fluorescent protein, thereby altering the fluorescence property of the modified fluorescent protein;
(C) reacting the resultant various fusion proteins with a factor affecting the conformation of any of the functional molecules; and
(D) screening a fusion protein exhibiting a change in the fluorescence property by the reaction of the step (C), as a biosensor protein, from the various fusion proteins.
[2] The method described in [1], wherein the fluorescence property is fluorescence intensity.
[3] A biosensor protein comprising (1) and (2) below:
(1) a modified fluorescent protein which is obtained by cleaving amino acid sequence of green fluorescent protein or its derivative in the vicinity of a hotspot amino acid residue which affects the fluorescence property, and modifying the structure of the green fluorescent protein or its derivative; and
(2) one or more functional molecules which are capable of transmitting their conformational changes to the modified fluorescent protein to cause a conformational change of the modified fluorescent protein, thereby altering the fluorescence property of the modified fluorescent protein.
[4] The biosensor protein described in [3], wherein the fluorescence property is fluorescence intensity.
[5] The biosensor protein described in [3] or [4], wherein the hotspot amino acid residue corresponds to the 148th amino acid of amino acid sequence of green fluorescent protein.
[6] The biosensor protein described in [3] or [4], wherein the hotspot amino acid residue corresponds to the 94th amino acid of amino acid sequence of green fluorescent protein.
[7] The biosensor protein described in [3] or [4], wherein the hotspot amino acid residue corresponds to the 96th amino acid of amino acid sequence of green fluorescent protein.
[8] The biosensor protein described in [3] or [4], wherein the hotspot amino acid residue corresponds to the 222nd amino acid of amino acid sequence of green fluorescent protein.
[9] A biosensor protein comprising (1) and (2) below:
(1) a modified green fluorescent protein having the following amino acid sequences (a) and (b) in this order from the N terminus:
(a) an amino acid sequence of X-th to 238th position of green fluorescent protein; and
(b) an amino acid sequence of 1st to Y-th position of green fluorescent protein,
(where X is an arbitrary number from 148 to 150, and Y is an arbitrary number from 140 to 147); and
(2) one or more functional molecules which are capable of transmitting their conformational changes to the modified green fluorescent protein to cause a conformational change of the modified green fluorescent protein, thereby altering the fluorescence property of the modified green fluorescent protein.
[10] A biosensor protein comprising the following sequences (a) to (h) sequentially from the N terminus:
(a) an amino acid sequence containing methionine (linker X);
(b) myosin light chain kinase protein or a partial amino acid sequence thereof;
(c) an amino acid sequence (linker Y) for linking the above sequence (b) and the following sequence (d);
(d) an amino acid sequence from X-th to 238th position of green fluorescent protein (where X is an arbitrary number from 148 to 150);
(e) an amino acid sequence for linking the above sequence (d) and the following sequence (f);
(f) an amino acid sequence from 1st to Y-th position of green fluorescent protein (where Y is an arbitrary number from 140 to 147);
(g) an amino acid sequence (linker Z) for linking the above sequence (f) and the following sequence (h); and
(h) calmodulin protein or a partial amino acid sequence thereof.
[11] A biosensor protein comprising (1) and (2) below:
(1) a modified green fluorescent protein having the following amino acid sequences (a) and (b) in this order from the N terminus:
(a) the amino acid sequence from the 149th to 238th position of green fluorescent protein, and
(b) the amino acid sequence from the 1st to 144th position of green fluorescent protein; and
(2) one or more functional molecules which are capable of transmitting their conformational changes to the modified green fluorescent protein to cause a conformational change of the modified green fluorescent protein, thereby altering the fluorescence property of the modified green fluorescent protein.
[12] A biosensor protein comprising the following sequences (a) to (h) sequentially from the N terminus:
(a) an amino acid sequence containing methionine (linker X);
(b) myosin light chain kinase protein or a partial amino acid sequence thereof;
(c) an amino acid sequence (linker Y) for linking the above sequence (b) and the following sequence (d);
(d) the amino acid sequence from the 149th to 238th position of green fluorescent protein;
(e) an amino acid sequence for linking the above sequence (d) and the following sequence (f);
(f) the amino acid sequence from the 1st to 144th position of green fluorescent protein;
(g) an amino acid sequence (linker Z) for linking the above sequence (f) and the following sequence (h); and
(h) calmodulin protein or a partial amino acid sequence thereof.
[13] The biosensor protein described in any one of [3]-[9], and [11], wherein the functional molecules are calmodulin protein or a partial amino acid sequence thereof, and myosin light chain kinase protein or a partial amino acid sequence thereof.
[14] A biosensor protein comprising the following sequences (a) to (h) sequentially from the N terminus:
(a) Met-Gly-Thr or Met-Val-Asp (linker X);
(b) a partial amino acid sequence of myosin light chain kinase protein (Serxe2x80x94Ser-Argxe2x80x94Arg-Lys-Trp-Asn-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu-Serxe2x80x94Ser) [SEQ ID NO:6]
(c) Leu-Glu (linker Y);
(d) the amino acid sequence from the 149th to 238th position of green fluorescent protein;
(e) Glyxe2x80x94Gly-Thr-Glyxe2x80x94Gly-Ser (linker amino acid sequence) [amino acid 117 to 122 of SEQ ID NO:8]
(f) the amino acid sequence from the 1st to 144th position of green fluorescent protein;
(g) Gly-Thr-Arg or Thr-Arg (linker Z); and
(h) the amino acid sequence from the 2nd to 148th position of rat calmodulin protein.
[15] A biosensor protein comprising the following sequences (a) to (c) in this order from the N terminus:
(a) the amino acid sequence from the 1st to 144th position of green fluorescent protein;
(b) one or more functional molecules which are capable of transmitting their conformational changes to the modified green fluorescent protein to cause a conformational change of the modified green fluorescent protein, thereby altering the fluorescence property of the modified green fluorescent protein; and
(c) the amino acid sequence from the 149th to 238th position of green fluorescent protein.
[16] The biosensor protein described in [15], wherein the functional molecules are calmodulin protein or a partial amino acid sequence thereof, and myosin light chain kinase protein or a partial amino acid sequence thereof.
[17] A biosensor protein comprising the following sequences (a) to (g) sequentially from the N terminus:
(a) the amino acid sequence from the 1st to 144th position of green fluorescent protein;
(b) an amino acid sequence (linker A) for linking the above sequence (a) and the following sequence (c);
(c) calmodulin protein or a partial amino acid sequence thereof;
(d) an amino acid sequence (linker B) for linking the above sequence (c) and the following sequence (e);
(e) myosin light chain kinase protein or a partial amino acid sequence thereof;
(f) an amino acid sequence (linker C) for linking the above sequence (e) and the following sequence (g); and
(g) the amino acid sequence from the 149th to 238th position of green fluorescent protein.
[18] A biosensor protein comprising the following sequences (a) to (g) sequentially from the N terminus:
(a) the amino acid sequence from the 1st to 144th position of green fluorescent protein;
(b) Gly-Thr-Arg (linker A);
(c) the amino acid sequence from the 2nd to 148th position of rat calmodulin protein;
(d) Gly-Thr or Gly-Thr-Gly-Ser-Glyxe2x80x94Gly-GlY-Ser (linker B) [SEQ ID NO:17];
(e) a partial amino acid sequence of myosin light chain kinase protein Serxe2x80x94Ser-Argxe2x80x94Arg-Lys-Trp-Asn-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu-Serxe2x80x94Ser) [SEQ ID NO:6];
(f) Thr-Ser (linker C); and
(g) the amino acid sequence from the 149th to 238th position of green fluorescent protein.
[19] A biosensor gene encoding the biosensor protein described in any one of [3]-[18].